Diethylstilbestrol-Induced Mouse Hypospadias: “Window of Susceptibility”

image pf hypospadias in human fetal penises

Defining a DES “programming window”

2016 Study Abstract

Hypospadias, an abnormality affecting the penile urethra, is one of the most prevalent congenital malformations afflicting human males. The morphology of hypospadias is markedly different in humans versus mice reflecting substantial differences in penile development in humans and mice. Estrogens such as diethylstilbestrol (DES) elicit mouse penile malformations, but the types of penile abnormalities differ depending on whether DES treatment is prenatal or neonatal.

A thorough investigation of the effects of DES over a wide age range of treatment may

  • elucidate the morphogenetic mechanisms involved in generating abnormal penile morphology and hypospadias
  • and reveal those penile elements more (or less) sensitive on a temporal basis to developmental exposure to DES.

Such an approach may also explain why certain effects of DES elicited and expressed during development resolve to normality in adulthood.

To define the actual “window of susceptibility” to the adverse effects of DES, pregnant mice and their neonatal pups were injected subcutaneously with 200ng/gbw DES every other day

  • from embryonic day 12 to 18 (DES E12-E18),
  • postnatal day 0 to 10 (DES P0-P10),
  • embryonic day 12 to postnatal day 10 (DES E12 to P10),
  • postnatal day 5 to 15 (DES P5 to P15),
  • and postnatal day 10 to 20 (DES P10 to P20).

Aged-matched controls received sesame oil vehicle. After euthanasia at 10, 15, 20 and 60 days, penises were analyzed by gross morphology, histology and morphometry.

Penises of all 5 groups of DES-treated mice were reduced in size, which was confirmed by morphometric analysis of internal penile structures, and are presumably mediated via signaling through estrogen receptors alpha and/or beta (ERα and ERβ), which have been previously detected in all of the structures affected by DES.

The most profound effects were seen in the DES E12-P10, DES P0-P10, and DES P5-P15 groups, thus defining a DES “programming window”.

For all parameters, DES treatment from P10-P20 showed the most mild of effects.

Adverse effects of DES on the MUMP cartilage and erectile bodies observed shortly after the last DES injection reverted to normality in the DES P5-P15, but not in the E12-P10 and P0-P10 groups, in which MUMP cartilage and erectile body malformations persisted into adulthood, again emphasizing a “window of susceptibility” in the early neonatal period.


  • Full study (free access) : Diethylstilbestrol-Induced Mouse Hypospadias: “Window of Susceptibility”, Differentiation, NCBI PubMed PMC4803596, 2016 Jan 20.
  • Scanning electron micrographs of human fetal penises at 7 and 10 weeks of gestation. In (A) note the prominent urethral groove. In (B) the edges of the urethral groove are fusing in the midline to form the urethra, but the distal urethral groove is still widely open. Featured image credit PMC4803596/figure/F3.

Hypospadias in DES grandsons : a cohort study

image of grandsons

Hypospadias: a transgenerational effect of diethylstilbestrol ?

2002 Study Abstract

Transgenerational effects of diethylstilbestrol (DES) have been reported in animals, but effects in human beings are unknown. Alerted by two case reports, we aimed to establish the risk of hypospadias in the sons of women who were exposed to DES in utero.

We did a cohort study of all sons of a Dutch cohort of 16284 women with a diagnosis of fertility problems. We used a mailed questionnaire assessing late effects of fertility treatment to identify boys with hypospadias. We compared the prevalence rate of hypospadias between boys with and without maternal DES exposure in utero.

16284 mothers (response rate 67%) reported 8934 sons. The mothers of 205 boys reported DES exposure in utero. Four of these children were reported to have hypospadias. In the remaining 8729 children, only eight cases of hypospadias were reported (prevalence ratio 21.3 [95% CI 6.5-70.1]). All cases of hypospadias were medically confirmed. Maternal age or fertility treatment did not affect the risk of hypospadias. Children conceived after assisted reproductive techniques such as in-vitro fertilisation were not at increased risk of hypospadias compared with children conceived naturally (1.8, 0.6-5.7).

Our findings suggest an increased risk of hypospadias in the sons of women exposed to DES in utero. Although the absolute risk of this anomaly is small, this transgenerational effect of DES warrants additional studies.


  • Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study, Lancet, NCBI PubMed PMID: 11943257, 2002 Mar 30.
  • Featured image Donna Borzyskowski.

DES and third-generation hypospadias

image of third-generation hypospadias

Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study

2011 Study Abstract

Prenatal diethylstilbestrol (DES)-exposed mice have raised the suspicion of a transgenerational effect in the occurrence of genital malformation in males. This nationwide cohort study in collaboration with a French association of DES-exposed women studied 529 families and showed that a significant proportion of boys born to DES daughters exhibited hypospadias with no other molecular defects identified.

Although the role of fetal androgens is crucial to male genital development during the first trimester of pregnancy, defects in the synthesis or molecular action of testosterone are rare in isolated hypospadias (Hypospadias may be a multifactorial defect arising from genetic, hormonal, and environmental factors). It has been hypothesized that changes in androgen/estrogen balance due to endogenous or exogenous hormonal factors during the critical period of penile and urethral development contribute to this malformation.

Men who were exposed in utero to diethylstilbestrol (DES), a synthetic estrogen, may exemplify the effects of environmental chemicals with endocrine-disrupting activity on genital development (DES was prescribed for pregnant women from the late 1930s to the 1970s in the mistaken belief that it would prevent miscarriage or premature birth). Unfortunately, DES was found to be not only ineffective but also harmful. Daughters born from DES-related pregnancies often show abnormalities in the Müllerian structures and have elevated risks of peripubertal vaginal and cervical clear-cell adenocarcinoma, fertility problems, ectopic pregnancies, miscarriages, and premature births (The risk of reproductive tract abnormalities also appears to be increased for DES sons, who may present hypoplastic testis, epididymal cysts, cryptorchidism, or hypospadias).

After several studies in animals, a question emerged as to whether the harmful effects of DES can be “transmitted” to subsequent generations. Newbold and colleagues reported an increased incidence of reproductive tract tumors in male and female descendants of mice developmentally exposed to DES. In the human, Klip et al. reported an increased risk of hypospadias in sons of DES daughters in a cohort of women diagnosed with fertility problems. Other studies either confirmed or questioned these results. However, the direct implication of DES in the occurrence of hypospadias remains debatable, since many other uncontrolled factors, especially environmental and genetic, are implicated in this malformation. We studied the prevalence of hypospadias in the grandsons of DES-treated and -untreated women and ruled out other environmental and genetic factors that could have been associated with the malformation in these patients.

A nationwide cohort study was conducted in collaboration with a French association of DES-treated women (HHORAGES Association). The reason the women joined this association was not the presence of hypospadias in the second or third generation but rather psychological disturbances, vaginal and cervical clear-cell adenocarcinomas, miscarriages, and other abnormalities. Five hundred twenty-nine families were included. All of the second- and third-generation offspring were accounted for and included in the study. No one declined to participate. DES exposure was reported in 1,000 out of 1,180 pregnancies. The featured image details the patient groups. The clinical diagnosis of hypospadias was standardized and based on a detailed operative report or direct clinical examination by a pediatrician and/or urologist. The malformation was characterized as severe (proximal, penoscrotal) or nonsevere (glandular, subcoronal, distal, midshaft). Each mother with a hypospadiac son was contacted and responded to a short questionnaire validated in Europe for data collection (no. QLK4-1999-01422) to determine whether other occupational exposure had occurred during the pregnancy. To exclude a defect of the androgen pathway, we performed molecular analysis of the genes known to be associated with hypospadias such as androgen receptor (AR), 5α reductase (srd5A2), and MAMLD1 genes in DNA from peripheral blood. The local university hospital ethics committee approved this study (ID RCB No. 2008-A00781-54), and each patient gave informed consent through the Hhorage Association.

The prevalence of hypospadias was low in boys unexposed to DES in utero (0/180), whereas it was high in the in utero–exposed boys (3.57%, 16/448, P=.02). In the third generation, the prevalence of hypospadias in boys born to DES daughters was high when compared with boys born to unexposed parents (8.2%, n = 8/97 vs. n = 0/360; P<.001). The hypospadiac patients of the second and third generations were not related. The results are summarized in the featured image.

Neither mutations nor polymorphisms of the AR and MAMLD1 genes were found among hypospadiac boys of the third generation. Only one polymorphism of the srd5A2 gene was detected (A49T) in a boy. The mothers of the third-generation affected boys indicated little environmental or occupational exposure to endocrine-disrupting chemicals during pregnancy (no professional activity, n = 2; sales clerk in a food or clothing shop, n = 3; office worker, n = 3), and such exposure was therefore unlikely to have contributed to the occurrence of hypospadias. Two mothers of hypospadiac sons exhibited hypoplastic or bifid uterus.

The main effect of DES is profound disturbance in the androgenic/estrogenic balance of animal and human fetuses since it has both estrogenic and antiandrogenic actions by competing with natural androgens for the ligand-binding domain of the androgen receptor. In utero exposure to DES during the critical period of reproductive tract development is known to induce genital malformation in mice. In utero–exposed sons show greater risks of structural urogenital abnormalities like hypospadias, epididymal cysts, micropenis, and cryptorchidism. The present study reinforces these data with a prevalence of hypospadias greater than 3%, although it should be noted that the second-generation population included only 180 controls from the same families since this series was specifically designed to study the third-generation boys.

More interesting is the hypothesis of a transgenerational effect of DES. Animal studies first suggested that DES might increase transgenerational susceptibility to malignant tumors of the female reproductive tract, presumably by damage to germ cells and abnormal imprinting. In human beings, DES exposure may also lead to permanently altered germ cells. The suggestion of a transgenerational effect of DES in human beings was based on the observation of a high prevalence of hypospadias, particularly with severe phenotypes, in the sons of women exposed to DES in utero.

But variations in the definition of the control population may explain the wide range of odds ratios reported in the literature to date. Palmer et al. reported a prevalence 6 times higher than that of Klip et al. The present study, which shows a high prevalence of hypospadias of various severities in the third generation, tried to limit this bias and included DES-free pregnancies and DES-exposed pregnancies from the same families. Nevertheless, two limitations should be noted:

  1. DES-exposed women without problems were not included;
  2. and the fertility status of the exposed and non-exposed couples, the age at pregnancy, and the parity for each women were variable, and this may have hidden fertility problems or greater use of contraception.

The low fertility rates of the DES sons may also be explained by other findings, such as severe psychotic disorders or oligospermia in cases of hypospadias with additional defects.

We did not identify any genetic or environmental factors that would have explained the hypospadias in DES grandsons. Our results thus raise the question of the mechanism through which DES causes adverse effects in subsequent generations. The frequency of transmission both observed in our series of hypospadiac grandsons and previously reported in generations examined for various disease states secondary to DES exposure is particularly high. This frequency of a transgenerational phenotype is such that a mutational event involving the DNA sequence could not be implicated. DES-induced changes in epigenetic background and alteration of DNA methylation could be significant factors in the susceptibility to disease development. The primordial germ cells undergo demethylation during migration and early colonization of the embryonic gonad, followed by remethylation starting at the time of sex determination in a sex-specific manner. The pregnant mother’s exposure to DES at the time of fetal sex determination appears to be sufficient to alter the remethylation of the germ line in the male fetus and permanently reprogram the imprinted pattern of DNA methylation. The transmission of multigenerational DES effects would thus occur through the paternal lineage. But our findings indicated that most of the third-generation hypospadiac boys were born to DES daughters. This agreed with previous studies (although paternal transmission of DES effects is not excluded). Epigenetic changes in the AR gene, transmitted through the DES daughter, could explain such a finding since the antiandrogen effect of DES is known to modify the phosphorylation level of AR.

The association between hypospadias in grandsons and uterine abnormalities in their mothers suggests other hypotheses for the transgenerational mechanisms of DES. First, DES daughters may have displayed disturbed hormonal balance during their reproductive life or placental malfunction that might have interfered with the genital development of a male fetus. Second, the estrogen receptor gene ERα and estrogen-responsive genes that contribute to the development of both female internal genitalia and hypospadias may also be involved since ERα is implicated in the induction of abnormalities after DES exposure. Last, the genes involved in the structural differentiation of both the female and male reproductive tracts may be altered by DES exposure. DES has been reported to delay expression of HOXa family genes during Müllerian duct development. DES could also interfere with HOX gene expression during penile formation.

For many authors, DES is an experimental environmental xenoestrogen. Despite the bias that could not be fully eliminated and the difficulty of extrapolating the risks of exposure (no monotonic dose-response relationship, varying effects depending on the timing of exposure in the developing organism, manifestations delayed until later in life), this clinical study strengthens the suspicion of the transgenerational effects of environmental endocrine disruptors.


  • Full text (free access) : Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study, CHU Montpellier and Universite Montpellier, France, hhorages, February 23, 2011.
  • Featured image : Detailed patient groups included in the study. In the second generation, the phenotype of affected boys was isolated hypospadias in all cases, severe in 12 cases (posterior or penoscrotal), and not severe in four cases (mild or anterior). In the third generation, the hypospadias was severe in five cases and not severe in three cases. Bilateral cryptorchidism was present in one case. *The size of the population was under 1,000, and the prevalence of hypospadias of about 1/1,000, as seen in the general population, could not be represented.

Hypospadias: a transgenerational effect of diethylstilbestrol?

image of des grandson

Is there an increased risk of hypospadias for DES grandsons?

2006 Study Abstract

In 2002, an increased risk of hypospadias was reported for sons of women exposed to diethylstilbestrol (DES) in utero, suggesting transgenerational effects of DES. The aim of this study was to further assess the association between parental DES exposure and hypospadias in a case-referent study.

Cases with hypospadias were retrieved from the hospital information system. Referents were recruited via the parents of cases. Both parents completed postal questionnaires. Associations were estimated by odds ratios (OR) with 95% confidence intervals (CI). Additionally, conditional logistic regression analyses were performed for a matched subset of parents.

The final database included 583 cases and 251 referents. In the initial analyses, an indication was found for an increased risk of hypospadias when mothers were exposed to DES in utero: OR=2.3 (95% CI 0.7-7.9). Conditional logistic regression resulted in a stronger risk estimate: OR=4.9 (95% CI 1.1-22.3). Paternal exposure to DES did not increase the risk.

The results confirm an increased risk of hypospadias when mothers were exposed to DES in utero. However, the excess risk appears to be of much smaller magnitude than in the 2002 study. Further research on the potential health risks for the third generation is of great importance.


  • Full text (free access) : Hypospadias: a transgenerational effect of diethylstilbestrol?, Human reproduction (Oxford, England), NCBI PubMed, PMID: 16293648, 2006 Mar.
  • Featured image credit Todd Cravens.